Defining molecular pathways that are dysfunctional in autism and autistic spectrum disorders (ASDs) is key to understanding their pathogenesis and developing future therapeutics. In Alzheimer's and Parkinson's Disease, identification of single gene mutations in the 5-10% of genetic cases have revealed core molecular pathways that are altered, including in the larger category of sporadic cases. A key question is whether a similar molecular pathway(s) will emerge for autism based on the recent identification of defined mutations and de novo genome copy number variations that account for 10-20% of ASDs. Here, we propose to take advantage of genetic manipulations available in Drosophila to explore the mechanisms by which the autism-associated endosomal protein, NHE9 (Na+/H+ exchanger 9), couples alterations in neuronal activity to modifications of synaptic connectivity. NHE9 is one of several newly identified genetic links that indicate abnormal endosomal trafficking and synaptic growth may predispose to autism. Work from my lab has recently characterized a synaptic growth and plasticity pathway in Drosophila where postsynaptic targets release retrograde signals in an activity-dependent manner, triggering synaptic maturation and growth. These growth signals are processed and regulated through trafficking in the presynaptic endosomal pathway. We hypothesize that mutations in NHE9 alter endosomal formation, or ligand-receptor association in endosomal compartments, through disruption of endosomal pH, leading to abnormal synaptic growth signaling and activity-dependent defects in brain wiring that might contribute to autistic behavior. Endosomes exhibit a progressive acidification from early endosomes (pH ~6.5) to lysosomes (pH ~4.5) that is essential for degradation and recycling of internalized ligand-receptor complexes and cell adhesion proteins. Together with the vacuolar V-ATPase, NHE9 is predicted to be the key molecular determinant of endosomal pH by allowing early signaling endosomes to remain relatively basic by transporting H+ ions out of this compartment, where ligand-receptor pairs can remain attached and transmit synaptic growth signals. We will determine if endosomal trafficking defects are present in NHE9 mutants, and whether NHE9 activity may be linked to other ASD mutants that include synaptic cell adhesion proteins like Neurexin and Neuroligin. The generation of new contact sites is likely to require removal or recycling of surface Neurexin and Neuroligin through the endosomal system, a process that may require NHE9. Linking a common synaptic defect in these seemingly unrelated proteins may reveal a conserved molecular pathway that is dysfunctional in autism.